Primary structure of myohemerythrin from the annelid Nereis diversicolor.

The metal-free form of Nereis diversicolor myohemerythrin was purified from whole animal extracts by trichloroacetic acid precipitation and ion exchange chromatography. The amino acid sequence of myohemerythrin has been determined. The protein is composed of 120 residues, possesses an unblocked N-terminus and is devoid of cysteine residues. It bears 62% sequence identity with Themiste zostericola myohemerythrin, the only other member of this subfamily sequenced to date. Within the family of hemerythrins, homology is particularly high in the segments involved in the binding of the two iron atoms and in the beta-turn-rich N-terminal segment.

[Homologies between hemerythrins of sipunculids and cadmium-binding metalloprotein (MP II) from a polychaete annelid, Nereis diversicolor]. / Homologies entre les hémérythrines des sipunculiens et une métalloprotéine complexant le cadmium (MP II) d'une annélide polychète, Nereis diversicolor.

The determination of the first 33 amino acids of the Cd-binding-protein (MP II) of Nereis diversicolor (Annelida, Polychaeta) shows a homology of 79 and 61% with 2 respiratory proteins of sipunculids, respectively the myohemerythrin and the hemerythrin. The positive reaction obtained by immunocytochemistry over the hemerythrocytes of Sipunculus nudus using antibodies raised against MP II and the presence of iron on the MP II reinforce this similarity.

Polypeptides related to mammalian procholecystokinin in the brain of an invertebrate, a marine worm, Nereis diversicolor: evidence from in ovo translation of mRNA.

Total mRNA extracted from brain of a sea worm Nereis diversicolor (Annelida, Polychaeta) were injected into Xenopus oocytes. The in ovo-translated polypeptides were analyzed through electrophoresis of immunoprecipitated products or Western blotting techniques. Some of these polypeptides cross-reacted antibodies elaborated against three mammalian (human and rat) procholecystokinin (pro-CCK) sequences. Polypeptides of 15, 64, and 70 kDa were determined, but two families of less obvious products, whose molecular masses were between 27-34 kDa and 46-60 kDa, appeared. Furthermore, the Western blotting technique also showed a cross-reaction between some of the polypeptides (64 and 70 kDa) extracted from brains and the antibodies used. From this work, evidence is given of the presence of epitopes shared by cellular polypeptides extracted from the brain, in ovo-translated products and mammalian pro-CCK. Furthermore, these data suggest the existence of a CCK precursor in the brain of N. diversicolor.

Amino acid sequence of polypeptide chain IIB of extracellular hemoglobin from the polychaete Tylorrhynchus heterochaetus.

The giant extracellular hemoglobin from the polychaete Tylorrhynchus heterochaetus consists of two types of subunits: a "monomeric" chain (chain I) and a disulfide-bonded trimer of chains IIA, IIB, and IIC. The complete amino acid sequence of chain IIB was determined. This chain has 148 amino acid residues and a molecular weight of 17,236 including a heme group. Of the residues in chain IIB, 74 (50%) and 34 (30%) were found to be identical with those in the corresponding positions in Tylorrhynchus chains IIC and I, respectively (Suzuki, T., Furukohri, T., and Gotoh, T. (1985) J. Biol. Chem. 260, 3145-3154). Marked differences were found between the chains of Tylorrhynchus and Lumbricus in the COOH-terminal regions. Significant differences were predicted between the monomeric chain I and the "trimeric" chains (IIB and IIC) in the hydropathy profiles and alpha-helical contents.

Subunit structure of extracellular hemoglobin from the polychaete Tylorrhynchus heterochaetus and amino acid sequence of the constituent polypeptide chain (IIC).

Tylorrhynchus cyanomethemoglobin reduced with dithiothreitol was separated by chromatofocusing into four heme-containing polypeptide chains (I, IIA, IIB, and IIC) and a non-heme chain (N). The molecular weights of chains IIA-C and N were confirmed to be the same by polyacrylamide gel electrophoresis in sodium dodecyl sulfate on a 10-20% gradient gel. The molecular weight of chain IIC was determined to be 17,415 (including heme) from the amino acid sequence. Chain N constitutes less than 5% of the total protein and has the same NH2-terminal sequence, suggesting that it is derived from chain IIA during the isolation procedure. Tylorrhynchus hemoglobin consists of two types of subunit with molecular weights of 16,327 (chain I) and approximately 50,000, and the latter splits into chains IIA-C in the presence of a reducing agent. On the basis of the accurate value obtained for the molecular mass of chain IIC, it was concluded that the subunit of approximately 50,000 daltons is a trimer of heme-containing chains IIA, IIB, and IIC linked by disulfide bonds. The cysteine residue at position 5 and the arginine at position 10 are conserved in the four heme-containing chains of Tylorrhynchus hemoglobin. The complete sequence of 149 residues of Tylorrhynchus chain IIC was determined. This sequence shows high homology with Tylorrhynchus chain I (Suzuki, T., Takagi, T., and Gotoh, T. (1982) Biochem. Biophys. Acta 708, 253-258) and Lumbricus chain AIII (Garlick, R. L., and Riggs, A. F. (1982) J. Biol. Chem. 257, 9005-9015).

Occurrence and coexistence in Nereis diversicolor O.F. Müller (Annelida Polychaeta) of substances immunologically related to vertebrate neuropeptides.

Numerous immunochemical and immunohistochemical studies have shown a wide distribution of several families of neuropeptides in invertebrates as well as vertebrates. There are relatively few data available for Annelida: Polychaeta. Therefore, we undertook an immunohistochemical investigation in the marine worm Nereis. Among the vertebrate type antibodies tested, those against met-enkephalin, LH-RH, vasopressin, oxytocin and ACTH had negative or only very slight effects. Slight to moderate reactions were obtained for VIP, SRIF, CRF, GRF, and leu-enkephalin. Moderate to very strong responses were found with anti-CCK/gastrin, -substance P, and -beta-MSH sera. Immunopositive reactions were usually observed in the entire CNS (except, until now, in neurosecretory cells, type II, in nuclei 20, and in nerve fibres located in the infracerebral neurohemal area). The immunoreactivity was, however, more or less abundant according to different CNS regions. For example, it appeared that the immunostaining for CRF is more important in the VNC while the leu-enkephalin family is more abundant in the brain (particularly in fuchsinophilic neurosecretory cells, type I, in nuclei 20). Moreover, several vertebrate type peptides (such as CRF/GRF and CCK/gastrin) may coexist in a single neurone. Several antisera may elicit a positive reaction in some specific area (for example, substance P in the nuchal organ; SRIF in oocytes; CCK/gastrin in the gastrointestinal tract). Nothing is known about the role of the different substances immunologically detected in Nereis. It is suggested that CCK/gastrin-, beta-MSH- and substance P-like materials transmit external stimuli to neurosecretory centres located in the caudal part of the brain.

Myoepithelial cells from a polychaete worm

Las células mioepiteliales que forman el proventriculo del polqueto Syllis spongiphila son interesantes morfológica y fisiológicamente. Cada célula consta de una región periférica, contractil y estriada, y una región central no contractil. La primera contiene uno o dos sarcómeros de una longitud de hasta 40 um. Cada filamento grueso (paramiosina) de unas 25 um de longitud, está rodeado de unos 20 miofilamentos finos. La región central encierra el núcleo, mitocondrias y vesículas llenas de un material cristalino. Experimentos con EGTA y "electron-probe analysis" demuestran que el Ca es el catión mas abundante en las vesículas, que son capaces de secuestrar calcio libre. Las células poseen un sistema tubular muy extenso, pero su retículo sarcoplásmico es rudimentario. Las células estas acopladas eléctricamente y reciben inervación excitadora (colinérgica) e inhibidora; se contraen al despolarizarse y se relajan al hiperpolarizarse, siendo capaces de producir potenciales de acción en los que la carga es llevada por iones de calcio. Sus canales par a calcio permiten el paso no solo de Ca, sino también de iones de Sr, Ba y Mn. en soluciones libres de calcio los potenciales de acción de Sr, Ba y Mn son de mayor duración que los Ca, pero solo los de Sr. producen contracción. experimentos con iones de Fe, Co y Ni suportan la idea de que la especifidad de los canales de Ca depende de su capacidad para distinguir las energias de hidratación de una población de cationes similares

High-performance liquid chromatographic separation of glycopeptides from Nereis cuticle collagen.

To facilitate the structural studies of invertebrate collagens, a sensitive and effective method was developed, using reverse-phase high-performance liquid chromatography for preparative isolation of the collagen subunits and their clostridial collagenase-derived peptides; the methods have been applied to Nereis cuticle collagen. The two subunits of denatured Nereis cuticle collagen, termed A and B, were initially separated by high-performance liquid chromatography. These polypeptides, with Mr of about 0.5 million, were each exhaustively digested with clostridial collagenase. The digest of the A subunit, which contains all of the uronic acid, was enriched for the uronic acid-containing glycopeptides by means of gel filtration. These glycopeptides were resolved into 23 major peaks, using reverse-phase HPLC, over a 5-h elution time, with an acetonitrile gradient (0-20%) containing 0.1% TFA. The amino acid composition data suggests that the peptides are of variable length, from 5 to 17 residues, while beta-elimination studies show that the uronic acid-containing moieties are all O-glycosidically linked to threonine residues, in the peptides examined. The amino acid sequence of one of the major glycopeptides was determined and found to be Gly-Hyp-Ala-Gly-Gly-Ile-Gly-Glu-Thr-Gly-Ala-Val-Gly-Leu-Hyp. The amino acid compositions of glycosylated and nonglycosylated peptides which had eluted, numbering about 100, showed a correspondence between hydrophobicity or hydrophilicity and emergence time from the column. We also found that the peptides most enriched in 4-hydroxyproline emerged earliest. These studies provide a foundation for elucidating the detailed structures of the large, unusual subunits of a well-characterized cuticle collagen.

Purification and characterization of oocyte vitellin from Perinereis cultrifera (polychaete annelid).

Vitellin was identified and purified from submature oocytes of Perinereis cultrifera by concanavalin-A - Sepharose and Ultrogel AcA 34 column chromatography. The yolk protein was defined as a glycolipoprotein with a relative molecular mass of 380000. Upon dissociation by sodium dodecyl sulphate, vitellin was shown to release five polypeptide subunits which ranged in relative molecular mass from 98000-16000. The purified vitellin was further characterized by amino acid analysis and its lipid and carbohydrate contents. The molecule contained about 5% carbohydrate and 16% lipid. Antiserum prepared against vitellin was shown to react with a component from the coelomic fluid of maturing females. Thus, it was suggested that in nereid annelids, considered phylogenetically and morphologically primitive, oocyte differentiation might depend upon the incorporation of a vitellogenin as in insects and many oviparous vertebrates.

The physico-chemical and functional properties of Abarenicola affinis affinis (Ashworth) extracellular haemoglobin (erythrocruorin).

The purified erythrocruorin from the Arenicolid polychaete, Abarenicola affinis affinis (Ashworth) has an S020,w of 57.5 S, and a molecular weight of 3.29 . 10(6) as determined by sedimentation equilibrium at pH 7.0. In the electron microscope it displays a two-tiered hexagonal array characteristic of other annelid erythrocruorins and chlorocruorins, with dimensions of approximately 25.5 nm diameter and 17.0 nm height. The isoelectric point of the molecule is at pH 4.63, and its amino acid composition and absorption spectrum are similar to those of other annelid erythrocruorins. The haem and iron contents are 2.77% and 0.23%, corresponding to minimum molecular weights of 22 250 and 24 800, respectively. In pH-induced dissociation studies, the erythrocruorin is found to be stabilized by Ca2+ up to pH 8.6, while in the presence of EDTA, dissociation begins at pH 7.8. In discontinuous gel electrophoresis, dissociated erythrocruorin gives subunits with molecular weights 263 000, 117 500, 68 400, 35 900 and 28 800, while, in detergent electrophoresis in the presence of 2-mercaptoethanol, polypeptide chains with molecular weights 13 700, 15 800, 24 700, 27 800 and 33 600 are obtained. Abarenicola erythrocruorin is also shown to bind oxygen cooperatively with a Hill coefficient h = 4.0 and P50 = 6.0 mmHg at pH 7.0 and 25 degrees C. Over the pH range of 7.0 to 7.8 the molecule lacks an alkaline Bohr effect.

The polypeptide composition of axoplasm and of neurofilaments from the marine worm Myxicola infundibulum.

1. Axoplasm from Myxicola contains two major polypeptides associated with neurofilaments, together with actin, tubulin and many minor polypeptide components. 2. Some of the minor polypeptides with molecular weights between 140,000 and 50,000 purify with neurofilaments under a variety of conditions and they appear to represent an integral part of the filament structure. 3. Peptide fingerprinting shows that the two major neurofilament polypeptides are almost identical. The fingerprint patterns from these major polypeptides share features with those obtained from the minor components. 4. Peptide fingerprinting has enabled us to propose a scheme for the main sites at which papain cleaves the major neurofilament polypeptides. In addition fingerprinting indicates how the minor components are related to the major polypeptides. 5. It is suggested that many of the minor neurofilament polypeptides could arise by proteolysis in vivo.

Subunit structure of erythrocruorin from the polychaete Tylorrhynchus heterochaetus.

Sodium dodecyl sulfate (SDS)-gel electrophoresis of erythrocruorin from the polychaete Tylorrhynchus heterochaetus revealed the presence of four subunits with molecular weights of 12,000, 22,000, 23,500, and 54,000 in a molar ratio of 6 : 1 : 2 : 3, respectively. The largest subunit dissociates into polypeptide chains of 13,500 molecular weight in the presence of mercaptoethanol, whereas each of the other subunits consists of a single polypeptide chain. Hemochromogen determination gave a minimum molecular weight of 26,500 per mol of heme group. A model of the subunit structure of the erythrocruorin molecule is proposed, composed of 144 subunits (252 polypeptide chains) possessing 144 heme groups. The molecular weight of the erythrocruorin was calculated to be 3.636 x 10(6).

[Determination of apparent mean mass of proteins associated with heme in the hemoglobin molecule of Arenicola marina (L.), Annelida, Polychaeta]. / Détermination de la masse apparente moyenne des protéines associées à l'hème dans la molécule d'hémoglobine d'Arenicola marina (L.), Annélide Polychète.

Protein and iron concentrations and maximum combined oxygen concentration were measured in the blood of the lugworm Arenicola marina. The calculated mean molecular mass of the heme-associated proteins was higher than that reported for known invertebrate and vertebrate intracellular hemoglobins. The difference is probably due to the presence of polypeptide chains not linked to heme groups in the extracellular annelid hemoglobins.

Isolation and characterization of a hemagglutinin from Amphitrite ornata, a polychaetous annelid.

Extracts of the marine polychaetous annelid, Amphitrite ornata, agglutinate rat, rabbit, chicken and human erythrocytes and in other work have been shown to inhibit the growth of Ehrlich ascites tumors in mice. Fractionation of extracts on Sephadex G-100 gave three active fractions with molecular weights of 30 000, 54 000 and 100 000. The 30 000 dalton fraction (B) was purified 72-fold by ammonium sulfate precipitation, gel filtration and preparative disc gel electrophoresis. The purified hemagglutinin, amphitritin, was homogenous on analytical disc gel electrophoresis at four different pH values and gave a sharp boundary in sedimentation velocity ultracentrifugation. The three fractions showed paralled specificity toward rat and chicken erythrocytes, the former giving the higher titer. The purified agglutinin was active toward human blood groups A, B and O and exhibited 4-fold higher activity toward group A. The hemagglutinin titer against rat red blood cells was lowered only by N-acetylgalactosamine, the terminal sugar residue of the group A determinant. None of the saccharides tested inhibited agglutination of chicken erythrocytes. Hemagglutinin activity was insensitive to dialysis or treatment with EDTA. The activity was not affected by digestion with trypsin or pronase, but was destroyed by phenol extraction. Analytical disc gel electrophoresis showed one protein band with high anodal mobility at pH 8.5, which was not affected by proteolytic enzymes but was removed by phenol. Activity was unaffected by heating at 70 degrees C for 30 min but was destroyed by similar treatemtn at 85 degrees C. Activity was at a maximum at pH 7-9 and decreased reversibly down to pH 4 at which point it was irreversibly inactivated. The higher molecular weight agglutinin (A1) could be dissociated to give amphitritin by treatment with 6M urea of precipitation in 55% (NH4)2SO4. This dissociation was not reversed by dialysis. Amphitritin is a glycoprotein with a molecular weight determined by gel filtration of 30 000 and by approach to equilibrium sedimentation of 32 000. Amino acid analysis showed a preponderance of aspartic and glutamic acids and relatively large amounts of glycine, proline, alanine, valine and cysteine. The carbohydrate moeity which represented 12.8% of the molecule, contained mannose, galactose, glucosamine and sialic acid. Amphitritin is the first hemagglutinin to be isolated from a polychaetous annelid.

Axoplasm chemical composition in Myxicola and solubility properties of its structural proteins.

The chemical composition of axoplasm extracted from the giant axon of Myxicola infundibulum has been analysed, and some of the factors which disperse its gel structure have been identified. 2. The axoplasm contains about 3-6% protein, and 0-12% lipid. It is isosmotic with sea water and has a pH near 7-0. 3. Inorganic ions in extracted axoplasm include: Na+, 13m-mole/kg wet wtl; K+, 280; Cl-, 24; Ca2+, 0-3; Mg2+, 3. 4. Free organic ions in axoplasm include: gly, 180 m-mole/kg wet st.; cysteic acid, 120; asp, 75; glu, 10; ala, 7; tau, 5; thr, 2; gln and ser, trace; homarine, 63; isethionate, 0. 5. The gel structure is dispersed by solutions containing 1--10 mM-Ca2+, because this ion activates an endogenous protease. The gel can also be dispersed without proteilysis by solutions containing 0-5 M-KCl, or 0-5 M guanidine hydrochloride, or 3-5 M urea, all of which break down neurofilaments. 6. It is argued that many aspects of the composition and dispersal properties of Myxicola axoplasm are similar to those in other axons.

The structure of annelid and mollusc hemoglobins.

The polypeptide chain composition and the chemical properties of several annelid hemoglobins and chlorocruorins are presented. In agreement with earlier studies on the hemoglobin from Arenicola cristata (Waxman, L. (1971) J. Biol. Chem. 246, 7318-7327), nearly all of the pigments which have been examined consist of one or more different disulfide-linked polypeptide chains of 13,000 to 16,000 daltons, and the heme-protein stoichiometry suggests that more than one polypeptide is associated with each heme. Except for the prosthetic group, there is no outstanding chemical difference between the chlorocruorins and the hemoglobins, nor is ther any apparent differnce between those hemoglobins which show cooperative oxygen binding properties and those which do not. The results suggest that all these hemoglobins have similar structures. On the other hand, the polypeptide chains of mollusc hemoglobins have molecular weights of greater than 220,000. Each polypeptide binds many heme groups. Thus, annelids use small polypeptide chains while molluscs use giant polypeptides to carry O2.